Cell Biology of Vasopressin-induced Water Channels

加压素诱导的水通道的细胞生物学

• 批准号: 10652774
• 负责人: Dennis Brown
• 金额: $ 52.7万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-20 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10652774
• 关键词: Actin-Binding Protein; Actins; Address; Affect; Animals; Biological Assay; Biological Products; Cell membrane; Cell physiology; Cell surface; Cells; Cellular biology; Clinical; Clinical Treatment; Clinical Trials; Complex; Congestive Heart Failure; Cytoskeleton; Data; Defect; Dehydration; Development; Disease; Down-Regulation; Duct (organ) structure; EGF gene; Electrolytes; Endocytosis; Epidermal Growth Factor Receptor; Epithelial Cells; Equilibrium; Erlotinib; Event; Exocytosis; F-Actin; Failure; Follow-Up Studies; Funding; Goals; Growth Factor Receptors; Health; Homeostasis; Hypertension; Hyponatremia; Inherited; Ion Channel; Kidney; Kidney Diseases; Knowledge; Ligands; Link; Liquid substance; Membrane; Modification; Molecular; Participant; Pathway interactions; Patients; Pharmaceutical Preparations; Phosphorylation; Physiology; Play; Process; Production; Proteins; Public Health; RPS6KA gene; Receptor Activation; Receptor Cross-Talk; Receptor Signaling; Recycling; Regulation; Regulatory Pathway; Renal function; Research; Retrieval; Ribosomal Protein S6 Kinase; Role; Signal Pathway; Signal Transduction; Specificity; Techniques; Translating; Urine; Vasopressin Receptor; Vasopressins; War; Water; Work; absorption; antidiuretic; aquaporin-2; arm; cohort; depolymerization; design; enzyme activity; equilibration disorder; ezrin; genetic regulatory protein; inhibitor; kidney cell; live cell imaging; novel; novel therapeutic intervention; novel therapeutics; pharmacologic; protein transport; receptor; superresolution microscopy; therapeutic target; trafficking; vasopressin resistant diabetes insipidus; water channel; water treatment

Because of a lack of safe, effective and specific treatments for disorders of water balance, the ultimate goal of our work is to identify novel intracellular pathways by which plasma membrane accumulation of the aquaporin 2 water channel (AQP2) in kidney principal cells can be normalized in the absence of a properly functioning vasopressin receptor (V2R) signaling pathway. The overall objective of this proposal is to dissect newly- identified aquaporin 2 (AQP2) trafficking and regulatory pathways in order to provide actionable, basic information that can be translated into cell-specific clinical advances for the treatment of these conditions. Nephrogenic diabetes insipidus (NDI) is caused by renal insensitivity to VP, and results in excessive urine production, whereas water retention, often a result of inappropriate VP secretion, occurs in conditions such as congestive heart failure. Aim 1 addresses the hypothesis that AQP2 itself directs its intracellular trafficking itinerary as an “active” cargo protein rather than a passive bystander. We propose that AQP2 “catalyzes” compartment-specific actin remodeling via direct and indirect interactions with different cohorts of actin- regulatory proteins. These include actin itself, the Arp2/3 actin remodeling complex during exocytosis, and the actin binding protein ezrin during endocytosis. Aim 2 will identify the cellular crosstalk mechanism(s) by which a tug-of-war between the vasopressin receptor (VP/V2R - positive action) and epidermal growth factor receptor (EGF/EGFR - negative effect) regulate AQP2 trafficking and water balance. We will first explore how EGF and other EGFR ligands inhibit the antidiuretic effect of VP by receptor activation and downstream signaling. Then we will ask how inhibition of the EGFR pathway results in VP-independent AQP2 phosphorylation and membrane accumulation by activating a non-canonical kinase, P90 ribosomal S6 kinase (RSK). Techniques central to the proposed work include the molecular characterization of protein interactions, advanced super resolution microscopy and live cell imaging, enzyme activity assays, expression of modified proteins in cultured epithelial cells, and whole animal physiology. Understanding novel cellular mechanisms of AQP2 regulation and defining more specific participants in intracellular signaling will open unexplored avenues of research into the regulation of fluid and electrolyte homeostasis. We expect that our data will allow the development of more selective and cell specific pharmacological strategies to regulate AQP2 trafficking in water balance disorders.

由于缺乏安全，有效和具体的水平疾病治疗，这是最终目标 我们的工作是识别新型细胞内途径，通过该途径，水通道蛋白2的质膜积累 在没有适当功能的情况下，可以将肾脏主要细胞中的水通道（AQP2）归一化 加压素受体（V2R）信号通路。该提案的总体目的是剖析新近 确定水通道蛋白2（AQP2）贩运和监管途径，以提供可行的，基本的 可以转化为细胞特异性临床治疗的信息。 肾原性糖尿病（NDI）是由对VP的肾脏不敏感引起的，导致尿液过量 生产，而保水（通常是由于VP分泌不适当）的结果，发生在诸如 充血性心力衰竭。 AIM 1解决了AQP2本身指导其细胞内贩运的假设 行程是“活跃”的货物蛋白，而不是被动旁观者。我们建议AQP2“催化” 特定于室特异性肌动蛋白通过直接和间接相互作用与不同的肌动蛋白的同类 调节蛋白。其中包括肌动蛋白本身，胞吐作用期间ARP2/3肌动蛋白重塑复合物，以及 内吞作用期间肌动蛋白结合蛋白EZRIN。 AIM 2将识别细胞串扰机制 加压素受体（VP/V2R-阳性作用）和表皮生长因子受体之间的拔河 （EGF/EGFR-负面效应）调节AQP2运输和水平衡。我们将首先探讨EGF和 其他EGFR配体通过受体激活和下游信号传导抑制VP的抗利尿作用。然后 我们将询问EGFR途径的抑制如何导致与VP无关的AQP2磷酸化和膜 通过激活非典型激酶P90核糖体S6激酶（RSK）积累。中心的技术 建议的工作包括蛋白质相互作用的分子表征，晚期超级分辨率 显微镜和活细胞成像，酶活性评估，在培养的上皮中修饰蛋白的表达 细胞和整个动物生理学。了解AQP2调节的新型细胞机制和定义 细胞内信号传导中更具体的参与者将为调节开放意外的研究途径 流体和电解质稳态。我们希望我们的数据将允许开发更有选择性和 细胞特定的药物策略，以调节水平疾病中AQP2贩运。